Insulin increases active sodium transport across isolated toad skin segments, and this response can be directly and continuously monitored by measuring the short circuit current. In this project it is intended to study tissue-binding and degradation of insulin by the toad skin preparation. It is also intended to evaluate the possible role of adenyl cyclase in the stimulatory action of insulin in this tissue. Preliminary studies showed: 1) graded responses to insulin concentrations varying from 10 to the minus 9th power M to 10 to the minus 7th power M; 2) similar responses with respect to time of onset and maximal effect, whether skins were exposed continuously or as briefly as 30 sec to insulin 10 to the minus 7th power M; 3) rapid disappearance of immunoreactive and TCA pricipitable insulin from the incubation medium; 4) impairment of responsiveness to insulin and vasopressin following exposure of skins to trypsin (1mg/ml) for 1 hr, but normal response to the stimulatory effect of theophylline. It is intended to perform the following additional studies: 1) determination of the effect of enzymes and other agents that alter the cell membrane surface upon responsivenss to insulin and upon the disappearance rate of insulin from the incubation medium; 2) evaluation of the effect of temperature upon insulin disappearance and biologic activity; 3) comparison of cyclic-AMP levels in incubation medium following exposure of skins to insulin, vasopressin and theophylline; 4) direct measurements of specific binding of I125-insulin by isolated toad skin epithelial cells; 5) measurements of cyclic AMP content of isolated epithelial cells following stimulation by insulin.